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"This is the first structure anyone has ever gotten of ACP complexed with one of the proteins, with which it interacts. We think it might serve as model for how ACP interacts with other proteins as well."

CHORI scientist Stuart Smith, PhD, and his colleagues, in a unique collaboration with the Structural Genomics Consortium at the University of Oxford, UK, report in the November issue of Chemistry & Biology the crystal structure of human phosphopantetheinyl transferase (PPTase) complexed with its its coenzyme A substrate and one of its acceptor proteins, the acyl carrier protein (ACP) component of the fatty acid megasynthase. Phosphopantetheine moieties derived from vitamin B5 play essential roles as components of several protein complexes in the translocation of reaction intermediates between catalytic centers.

"The role of the PPTase is to transfer the phosphopantetheine from coenzyme A to its acceptor proteins," explains Dr. Smith. "It's an unusual but essential posttranslational modification."

By analysis of the crystal structures of the PPTase complexed with coenzyme A and ACP and characterization of a panel of PPTase mutants, Dr. Smith and his colleagues were able to discover new and definitive details as to how PPTase actually binds coenzyme A and ACP, and then catalyzes the phosphopantetheinyl transfer reaction.

More significantly, Dr. Smith's analyses hinted at a potential answer to one of the most intriguing questions still under investigation about human PPTase.

"While microorganisms typically contain several PPTases, each specialized for servicing one of several different acceptor proteins," Dr. Smith explains, "in higher animals there are apparently 3 or 4 different enzymes that are all serviced by this single PPTase."

Since this surprising discovery of what Dr. Smith refers to as the promiscuous PPTase, which was made by his group a few years ago, researchers have been left wondering how the human PPTase recognizes each of the different acceptor proteins.

"There is a region in the PPTase that is well ordered in the absence of ACP but which becomes disordered when ACP is bound," explains Dr. Smith.

"This region is close to the ACP-binding site and while this is still quite speculative, we think that it may be utilized in recognizing some of the other proteins with which PPTase interacts."

Though still requiring further investigation to corroborate, such a suggestion hints at the first clues as to how PPTase interacts with so many different enzymes. In addition, the study not only provides researchers with a more complete understanding of how PPT functions in higher animals, but it also provides critical information to scientists engaged in drug design.

As Dr. Smith explains, the original interest in human PPTase came from the hope that it could be used as a drug target to inactivate fatty acid synthase, one of the phosphopantetheine-requiring enzyme systems.

"That idea was dropped when we discovered that humans have only the one PPTase," says Dr. Smith. "That meant that inactivation of the enzyme could potentially knock out several different metabolic pathways, not just the one that was being targeted."

While human PPTase is no longer a drug target, bacterial PPTase could still be utilized in designing antibacterial agents.

"Microbiologists are always looking for inhibitors to use as antibacterial agents," Dr. Smith explains. "It's important for them to know that any inhibitor they might design would be specific for the antibacterial PPTase - and not a human counter part."

Dr. Smith's analyses provide just that kind of essential information for microbiologists.

"It turns out that the mode of interaction of the human PPTase with ACP differs significantly from that of its bacterial counterparts," says Dr. Smith. "The interactions are more hydrophobic than ionic. This difference could be exploited to improve the specificity of putative antimicrobial agents that are targeted to bacterial PPTases."

In addition, Dr. Smith's CHORI team and collaborators at Oxford have recently solved the crystal structure of another of the component enzymes of the fatty acid megasynthase.

"The ultimate goal," says Dr. Smith, "is to provide a detailed structure of the entire megasynthase that could be exploited to develop and refine novel therapeutic agents that could find application in the treatment of obesity."